T regulatory (Treg) cells constitute 5-10% of CD4+ T cells in humans and rodents and constitutively express CD25, CD28, CTLA-4, GITR, CD62L, and 4-1BB, as well as the transcription factor FoxP3, which is involved in their development and function. IL-2 also appears to play an important role in Treg cell development and homeostatsis because animals deficient for IL-2 or components of its receptor develop T cell hyperproliferation and autoimmune diseases that can be corrected by adoptive transfer of Treg cells from naive animals. Similarly, a lack of signaling through CD28/CD80 interaction is associated with reduced number and functionality of Treg cells, suggesting that this receptor/ligand system plays an important role in the development and function of Treg cells.
Naturally arising CD4+CD25+FoxP3+ Treg cells are a distinct cell population of cells that are positively selected on high affinity ligands in the thymus and that have been shown to play an important role in the establishment and maintenance of immunological tolerance to self antigens. Deficiencies in the development and/or function of these cells have been associated with severe autoimmunity in humans and various animal models of congenital or induced autoimmunity.
Treg cells manifest their tolerogenic effects directly via cell-to-cell contact or indirectly via soluble factors. Although the suppressive mechanisms of these cells remain to be fully elucidated, blockade of IL-2 expression in effector T cells (Teff), physical elimination of Teff cells, induction of tolerogenic dendritic cells (DCs) via CTLA-4/B7 axis, and inhibition of Teff cells via TGF-β and IL-10 are some of the mechanisms that have been implicated to date. It also has been shown that reverse signaling through CTLA-4/CD80 into Teff cells plays an important role in their inhibition by Treg cells. Similarly, interactions between CTLA-4 on Treg cells and CD80 on DCs can result in reverse signaling and upregulation of the indoleamine dioxygenase enzyme that is involved in tolerance via the regulation of tryptophan metabolism.
In addition to their natural role in establishing and maintaining tolerance to self-antigens, Treg cells also have been shown to play a role in peripheral tolerance to foreign antigens induced by various immunomodulatory approaches. For example, it appears that Treg cells are the common denominator of mechanisms involved in peripheral tolerance to transplantation antigens, irrespective of the immunomodulatory approach used to achieve tolerance. Treg cells also have been implicated in immune evasion mechanisms by tumors and various pathogens.
The importance of Treg cells in establishing and maintaining tolerance to self-antigens and induced tolerance to foreign antigens has generated significant interest in methods for expanding Treg cells ex vivo for therapeutic purposes. See, e.g., Tang et al., 2004, J. Exp. Med. 199: 1455-65; Battaglia et al., 2005, Blood 105: 4743-48; Earle et al., 2005, Clin. Immunol. 115: 3-9; Godfrey et al., 2004, Blood 104: 453-61; Hoffmann et al., 2004, Blood 104: 895-903. Inasmuch as Treg cell development occurs via signaling through T cell receptors (TCR), CD28, and IL-2, methods of expanding Treg cells have focused on providing these three signals. See, e.g., Tang et al., supra; Godfrey et al., supra; Hoffmann et al., supra. For example, Tang et al., supra, reported that Treg cells could be expanded from nonobese diabetic (NOD) animals using stimulation with beads conjugated to anti-CD3 and CD28 antibodies in the presence of high doses of IL-2 (2000 IU/ml). Adoptive transfer of expanded TCR transgenic Treg cells specific for an auto-antigen prevented diabetes in an adoptive transfer model and reversed diabetes in newly diabetic NOD mice. A limited number of other expansion protocols based on this protocol have recently been developed with some success in expanding Treg cells from rodents and humans. See, e.g, Earle et al, supra; Godfrey et al., supra. For example, Godfrey et al. reported expansion of human Treg cells using a FcγRII (CD32) expressing cell line as an alternative to beads for fixing antibodies against CD3 and CD28 on the cell surface via Fc receptors. Almost all reported ex vivo expansion protocols are based on similar schemes and require the use of high doses of IL-2 to be effective.
Despite these advances, there remains a need for methods and compositions useful for expanding Treg cells ex vivo. There is a particular need for methods that do not require the use of a solid support. There also is a particular need for methods that do not require high doses of IL-2 for efficacy. There also is a need for methods and compositions useful for expanding Treg cells in vivo.
Type 1 diabetes remains a major cause of long-term morbidity and mortality in over one percent of population worldwide. Although insulin treatment and islet transplantation are currently the most effective therapeutic regimens, both of these approaches suffer from major limitations. Thus, there remains a need for methods of inducing islet-specific auto- , allo-, and xeno-tolerance for efficient and permanent treatment of Type 1 diabetes.
As discussed above, Treg cells play an important role in the control of self-reactive responses and in the establishment and maintenance of tolerance to foreign antigens. Treg cells, therefore, present an important therapeutic target for the prevention and treatment of various autoinunune diseases, including Type 1 diabetes, rejection of solid organs, tissues, stem cells, bone marrow cells, hematopoietic stem cells, and graft-vs-host disease (GVHD). There remains a need for methods for the controlled and deliberate expansion of Treg cells in vivo for the treatment of these conditions.